ORIGINAL PAPER

Direct electrochemistry of glucose oxidase and sensing glucoseusing a screen-printed carbon electrode modified with graphitenanosheets and zinc oxide nanoparticles

Chelladurai Karuppiah & Selvakumar Palanisamy &

Shen-Ming Chen & Vediyappan Veeramani &Prakash Periakaruppan

Received: 10 January 2014 /Accepted: 3 April 2014 /Published online: 24 April 2014# Springer-Verlag Wien 2014

Abstract We have studied the direct electrochemistry of glu-cose oxidase (GOx) immobilized on electrochemically fabri-cated graphite nanosheets (GNs) and zinc oxide nanoparticles(ZnO) that were deposited on a screen printed carbon elec-trode (SPCE). The GNs/ZnO composite was characterized byusing scanning electron microscopy and elemental analysis.The GOx immobilized on the modified electrode shows awell-defined redox couple at a formal potential of −0.4 V.The enhanced direct electrochemistry of GOx (compared toelectrodes without ZnO or without GNs) indicates a fastelectron transfer at this kind of electrode, with a heterogeneouselectron transfer rate constant (Ks) of 3.75 s−1. The fast elec-tron transfer is attributed to the high conductivity and largeedge plane defects of GNs and good conductivity of ZnO-NPs.Themodified electrode displays a linear response to glucose inconcentrations from 0.3 to 4.5 mM, and the sensitivity is30.07 μA mM−1 cm−2. The sensor exhibits a high selectivity,good repeatability and reproducibility, and long term stability.

Keywords Electrochemicalactivation .Graphitenanosheets .

ZnO nanoparticles . Glucose oxidase . Directelectrochemistry . Glucose biosensor

Introduction

The electrochemistry of redox active proteins (enzymes)has received substantial attention owing to their interest-ing electron transfer properties in biological systems,which has been vastly used for the application in bio-chemical and biophysical sciences [1]. In particular, thedirect electron transfer (DET) of redox active proteinsbetween electrode surface and redox active group has paidmuch notice due to their structural and biological activityfor the construction of biosensor and biofuel cells [2].Glucose oxidase (GOx) is one of the model redox activeflavin enzymes, which has been mostly used for theelectrocatalysis of glucose [3, 4]. The DET of GOx atbare electrodes is very difficult due to the complicatestructure and FAD redox active site of GOx that is deeplyburied in the protein cell [5]. Hence, the modified elec-trodes are utilized to promote the DET between the FADactive site of GOx and the electrode surface, includingcarbon nanotubes, graphene, metal nanoparticles, metaloxides, conducting polymers, ionic liquids, etc., [6–8].The carbon nanotubes and graphene composites havebeen extensively used for the fabrication of enzyme basedbiosensors due to their excellent conductivity, fast elec-tron transfer and high surface area [9]. Hence, the fabri-cation of novel materials has gained wider interest in theadvancement of redox electrochemical reaction of GOx onthe electrode matrix.

Fascinatingly, the electrochemical activation of carbonnanostructures has become more attractive and known for along time [10]. The activated carbon nanomaterials like graph-ite, carbon nanotubes and graphene have shown enhancedelectrochemical activity and fast electron transfer towardsthe several analytes, such as NADH, dopamine, hydrazine,and ascorbic acid [11, 12]. Graphite is an anisotropic carbon

Electronic supplementary material The online version of this article(doi:10.1007/s00604-014-1256-z) contains supplementary material,which is available to authorized users.

C. Karuppiah : S. Palanisamy : S.<M. Chen (*) :V. VeeramaniElectroanalysis and Bioelectrochemistry Lab, Department ofChemical Engineering and Biotechnology, National TaipeiUniversity of Technology, No.1, Section 3, Chung-Hsiao East Road,Taipei 106, Taiwan, Republic of Chinae-mail: smchen78@ms15.hinet.net

P. PeriakaruppanPost Graduate and Research Department of Chemistry, ThiagarajarCollege, Madurai 625009, Tamil Nadu, India

Microchim Acta (2014) 181:1843–1850DOI 10.1007/s00604-014-1256-z

material which has been widely used for the fabrication ofgraphite oxide, graphene oxide and graphene [13]. The graph-ite has highly reactive large edge planes with inert basalplane defects, which is more favourable for the activation ofgraphite [14]. Hitherto, different methods have beenemployed for the activation of graphite including the chem-ical activation and electrochemical activation [15, 16]. Theelectrochemical activation method is simple, green, environ-mental friendly and cost effective when compared to othermethods employed for the activation of graphite [14]. Overthe past years, the metal oxides have been widely employedfor the immobilization of GOx owing to their large surfaceactivity and rapid electron transfer ability [17]. Amongthem, zinc oxide (ZnO) is a special class metal oxide havingthe high isoelectric point (9.5), which is more favorable forthe immobilization of various redox active enzymes, such asGOx, horseradish peroxidase, cytochrome c, hemoglobinetc., [17–20]. The integration of activated graphite withZnO is relatively new and novel platform for the immobili-zation of GOx. To the best of our knowledge, the activatedgraphite or its composites have never been used as animmobilization matrix for the redox active proteins and notbeen reported yet in the literature.

In the present paper, the DET of GOx has been inves-tigated at electrochemically fabricated graphite nanosheets(GNs) and ZnO nanoparticles (ZnO) composite modifiedscreen printed carbon electrode (SPCE) for the first time.The GOx immobilized GNs/ZnO composite has beenfurther used for the construction of glucose sensor. Theselectivity, stability and practicability of the biosensorhave also been studied in detail.

Experimental

Chemicals

Raw graphite with an average diameter of about >20 μm wasobtained from Sigma to Aldrich (http://www.sigmaaldrich.com/taiwan.html). Screen printed carbon electrode waspurchased from Zensor R&D Co., Ltd., Taipei, Taiwan(http://www.zensor.com.tw/about/about.htm). Glucoseoxidase from Aspergillus Niger was obtained from SigmaAldrich (http://www.sigmaaldrich.com/taiwan.html). D (+)Glucose was obtained from Wako pure chemical industries(http://www.wako-chem.co.jp/english/labchem/). Zinc nitratehexahydrate (Zn (NO3)2. 6H2O) was obtained from Aldrich(http://www.sigmaaldrich.com/taiwan.html). The supportingelectrolyte was prepared by using 0.05 M Na2HPO4 andNaH2PO4 solutions in doubly distilled water. All thechemicals used in this work were of analytical grade and allthe solutions were prepared using doubly distilled waterwithout any further purification.

Apparatus

A computerized electrochemical workstation CHI 750a wasused for the cyclic voltammetry (CV) measurements.Scanning electron microscopy (SEM) was performed usingHitachi S-3,000 H electron microscope. An energy-dispersiveX-ray (EDX) spectrum was recorded using HORIBA EMAXX-ACT attached to Hitachi S-3,000 H scanning electron mi-croscope. A conventional three-electrode system was used forthe electrochemical experiments, a screen printed carbon elec-trode (active surface area =0.3 cm2) was used as a workingelectrode, a saturated Ag/AgCl as a reference electrode and aplatinum electrode as an auxiliary electrode. All measure-ments were carried out at room temperature in the inertatmosphere.

Fabrication of GNs/ZnO composite and immobilizationof GOx

The GNs and electrochemically activated SPCE were fabri-cated according to our previous method [21]. The fabricatedGNs modified SPCE was transferred to an electrochemicalcell containing 0.5 M (ZnNO3)2·6H2O and 0.1 M KNO3

solution, followed by applying a constant potential of−0.6 V for 200 s. Finally, ZnO was electrodeposited onto theGNs surface and the fabricated composite was named as GNs/ZnO composite. Similarly, ZnO modified electrode was alsoprepared by the same method without GNs.

For the immobilization of GOx, about 8 μL of GOx solu-tion (5mgmL−1) was drop-coated on the GNs/ZnO compositeand dried at room temperature. The obtained GNs/ZnO/GOxcomposite modified SPCE was gently rinsed a few times withdoubly distilled water to remove the loosely bound GOx. TheGNs/ZnO/GOx composite modified electrode was used forfurther electrochemical experiments and it was stored in pH 7solution at 4 °C when not in use.

Results and discussion

Characterization

Figure 1 shows the SEM images of ZnO/SPCE (a), electro-chemically fabricated GNs (b), and GNs/ZnO at lower (c) andhigher (d) magnifications. The SEM image of GNs clearlyreveals that the each graphite sheet edge is etched by electro-chemical pre-treatment method, leading to the successfultransformation of GNs. The obtained morphology of GNs isquite similar to previously reported chemically activated GNs[16]. The thickness of each GN is observed around 82±7 nm,which is quite lower than that of pristine graphite. As shown inFig. S1b, the observed surface of the pristine graphite sheetsare relatively smooth compared to that of GNs. The results

1844 C. Karuppiah et al.

clearly indicate that GNs have more edge plane defects withhigher surface area than pristine graphite (Fig. S1B). Thecracked surface of the electrochemically activated SPCE con-firms that the electrochemical pre-treatment greatly enhancesthe surface nature of SPCE (Fig. S1A). It can be seen fromFig. 1C to D that ZnO is uniformly deposited on the surface ofthe GNs surface without altering the morphology of the GNs.The ZnO is found uniform in size on the GNs. The observedmorphology and size of ZnO at SPCE are similar to that of ZnO(80±11) which is electrodeposited onto the GNs (Fig. 1a).The result further confirms that the morphology of the electro-deposited ZnO at SPCE and GNs is identical. We have alsoinvestigated the surface morphology of GNs/ZnO compositeafter the immobilization of GOx. It is evident from the Fig. S1Cto D that the dense clusters of GOx protein molecules arecovered on the whole surface of the GNs/ZnO composite.The result confirms that the GNs/ZnO composite possesseshigh affinity towards the immobilization of GOx.

The formation of ZnO on GNs is further confirmed by theelemental analysis. Figure 2a and b show the typical EDXprofiles of GNs and GNs/ZnO composite modified SPCE. Astrong and sharp elemental peak of carbon appears at 0.35 EVat GNs modified SPCE authenticating the pure carbon form ofGNs. That the GNs/ZnO composite modified SPCE showstwo new elemental peaks at 0.5 and 1.0 EV is attributed to thepresence of oxygen and metallic Zn respectively.

Direct electrochemistry of GOx

In order to evaluate the DETof GOx, CV was employed at thedifferent modified electrodes. Figure 3 shows the CVresponse

of bare SPCE/GOx (a), electrochemically activated SPCE/ZnO/GOx (b), SPCE/GNs/GOx (c), and SPCE/GNs/ZnO/GOx (d) in N2 saturated pH 7 solution at a scan rate of50 mV s−1. At bare SPCE/GOx, no significant electrochemi-cal signal of GOx appears owing to the very poor immobili-zation of GOx on the SPCE surface. A well defined redoxpeak response is observed at a formal potential of −0.4 V(Standard deviation for five electrodes is about 1.7 %) at GOximmobilized GNs/ZnO composite modified electrode. It isattributed to the presence of reversible electron transfer natureof the redox active center (FAD/FADH2) of GOx [22, 23]. Thehigh conductivity and large edge plane defects of GNs withgood electron communication capability of ZnO leads to thefast electron transfer of GOx [17]. Moreover, the excellentproperties of the GNs/ZnO composite are favorable for themore enzyme adsorption than GNs and electrochemicallyactivated SPCE/ZnO electrodes. The peak-to-peak separation(ΔEp) at GNs/ZnO/GOx is calculated as 35 mV, which issmaller than those obtained at SPCE/GNs/GOx/SPCE(48 mV) and activated SPCE/ZnO/GOx (50 mV) modifiedelectrodes. The obtained peak current of the redox couple ofGOx is relatively small at electrochemically activatedSPCE/ZnO/GOx and GNs/GOx modified electrodes com-pared to GOx immobilized GNs/ZnO composite. The ΔEp

at GOx immobilized GNs/ZnO composite is smaller thanthat of previously reported GOx immobilized modifiedelectrodes indicating the fast and reversible direct electrontransfer process of GOx [3, 22–26]. In addition, the peakcurrent intensities of the GOx redox couple at GNs/ZnOcomposite is about 2.5 and 5 folds higher than SPCE/GNsand activated SPCE/ZnO modified electrodes. The surface

Fig. 1 Typical SEM images ofZnO/SPCE (a), fabricated GNs/SPCE (b), and GNs/ZnO/SPCE atlower (c) and highermagnifications (d)

Direct electrochemistry of glucose oxidase and sensing glucose 1845

coverage concentration (Γ) of GOx at GNs/ZnO compositemodified electrode is calculated as 8.86 × 10−11 mol cm−2

using the Eq. 1.

Γ ¼ Q=nFA ð1Þ

Where a Q = total charge (5.2505 × 10−6 C), the n = numberof electrons transferred in the reaction (n=2), F = Faraday

constant (96,485.34 C mol−1) and A = active surface area ofthe electrode (0.307 cm2). The real active surface area of theGNs/ZnO modified electrode is calculated by CV using equa-tion (A=n2F2Ip.Γv/4RT) at 0.1 mM K4[Fe(CN)6]. 3H2O in0.1 MKCl solution at the scan rate of 50 mV s−1. The obtainedsurface coverage concentration is much higher than that GOximmobilized at Au nanoparticles/carbon paste (9.8×10−12 mol cm−2) [3] and multiwalled carbon nanotubes(2.86×10−12 mol cm−2) [27] modified electrodes. The highsurface coverage concentration and fast direct electron transferof GOx are attributed to the large surface area and high electroncommunication capability of the GNs/ZnO composite.

Effect of different sweeping scan rate and pH

The effect of different scan rate and pH at GOx immobilizedGNs/ZnO composite modified electrode was investigatedusing CV. Figure 4 shows the CVs of GNs/ZnO compositemodified electrode in N2 saturated pH 7 solution at differentsweeping scan rates (50 to 500 mV s−1). It can be seen that theanodic (Ipa) and cathodic (Ipc) peak currents increase linearlywith increasing the scan rates from 50 to 500 mV s−1. Thecathodic (Epc) and anodic (Epa) peak potentials are slightlyshifted with the slight increment of the peak to peak separation(ΔEp) at higher sweeping rates (>200 mV s−1). Moreover, theIpa and Ipc increase linearly with increasing scan rates from 50to 500 mV s−1 (inset), indicating that the redox reaction ofGOx at the GNs/ZnO composite electrode is a surface-confined reversible electron transfer process. According tothe Lavirons’ equation [28], the heterogeneous electron

Fig. 2 The EDX spectrum of SPCE/GNs (a), and SPCE/GNs/ZnO (b)

Fig. 3 The CV response of bare SPCE/GOx (a), electrochemicallyactivated SPCE/ZnO/GOx (b), SPCE/GNs/GOx (c), and SPCE/GNs/ZnO/GOx (d) in N2 saturated pH 7 solution at a scan rate of 50 mV s−1

Fig. 4 The cyclic voltammograms obtained at GOx immobilized GNs/ZnO modified electrode in N2 saturated pH 7 solution at different scanrates (50 to 500 mV s−1). Inset shows the corresponding linear plot for thedifferent scan rates vs. Ipa and Ipc

1846 C. Karuppiah et al.

transfer rate constant (Ks) for GOx immobilized at GNs/ZnOcomposite modified electrode is calculated as 3.75 s−1. Thecalculated Ks value is much higher than that of previouslyreported GOx immobilized multiwalled carbon nanotubes andgraphene modified electrodes [24, 29–31], which validatesthat GNs/ZnO composite facilitates the fast direct electrontransfer between the GOx and electrode.

Figure 5 shows the effect of pH on GOx (FAD/FADH2)redox couple at GNs/ZnO composite modified electrode invarious pH solutions at the scan rate of 50 mV s−1. It can beseen that a pair of well defined reversible redox peaks of GOxis observed in each pH solution. Both anodic and cathodicpeak potentials are shifted to negative and positive directionupon increasing and decreasing the pH and the maximumredox peak current response (Ip) is observed at pH 7 solution.The formal potential (E°) of GOx exhibits a linear dependenceover a wide pH range from pH 3 to 11 with a slope value of−61.2 mV/pH (R2=0.9921, inset of Fig. 5). This slope value isclose to the theoretical value of −58.6 mV/pH for equalnumber of protons (H+) and electrons (e−) involving electrontransfer process [9]. The result confirms that the direct elec-trochemical reaction of the redox couple at GOx is involvingan equal number of protons (H+) and electrons (e−). The directelectrochemistry of GOx at GNs/ZnO composite modifiedelectrode can be expressed by Eq. 2 [22, 32].

GOxþ FADþ 2 e− þ 2 Hþ↔GOx−FADH2 ð2Þ

Oxidation of glucose and reduction of O2

In order to evaluate the electrocatalytic activity of the GOxtowards glucose, CVs were recorded using GOx immobilizedGNs/ZnO composite modified SPCE. Figure 6a shows the

cyclic voltammetry response of composite modified electrodein O2 saturated PBS for different concentrations of glucose(0.3 to 6 mM) at the scan rate of 50 mV s−1. It can be seen thatthe oxidation peak current (Ipa) increases while the reductionpeak current (Ipc) decreases significantly upon increasing theconcentration of glucose form 0.3 mM to 6 mM. This is due tothe decrease in the O2 content of the solution as it is consumedduring the oxidation of glucose by the immobilized GOx onGNs [33]. The mechanism of glucose oxidation by GOx canbe expressed by Eq. 3 as reported previously [34].

GOx− FADð Þ þ Glucoseþ O2→GOx− FADð Þ þ Gluconolactoneþ H2O2 ð3Þ

The mechanism of oxygen reduction by GOx can be foundin electronic supplementary information (ESI). The Ipc linearlydecreases with the glucose concentration over the linear rangefrom 0.3 to 4.5 mM with a correlation coefficient of 0.9904.The equation of the calibration plots (Fig. 6b) are: I (μA)=9.0213+43.56 c (mM). From the fitted linear regression equa-tion, the sensitivity is calculated as 30.07 μAmM−1 cm−2. Thelimit of detection (LOD) is estimated as 0.07 mM (S/N=3).The blood glucose level in healthy humans is ranging from 4to 6 mM. Thus, the obtained linear range of glucose at GOximmobilized GNs/ZnO composite is more favorable for thedetermination of glucose levels in blood. Here it is essential toexplain the advantage of our biosensor compared to existingglucose biosensors, since carbon nanomaterials, metal nano-particles and metal oxides and their composites have been

broadly used for the construction of glucose biosensor [9, 12,17]. Hence, the analytical performance (linear range, sensitiv-ity, LOD and stability) of our biosensor has been comparedwith previously reported similar modified electrodes for theglucose detection and the comparative results are shown inTable 1. It is evident from Table 1 that our biosensor exhibitshigher sensitivity, stability along with fast heterogeneous elec-tron transfer constant (Ks) than previously reported GOximmobilized carbon nanotubes and reduced graphene oxidecomposites modified electrodes. The linear range and LOD ofour biosensor is comparable to aforementioned glucose bio-sensors. Most of the glucose biosensors reported in the liter-ature are based on carbon nanotubes, graphene and theircomposites modified electrodes. However, these modifiedelectrodes are still expensive and need more time for

Fig. 5 The CV response of GOx immobilized GNs/ZnO modified elec-trode in different pH solutions at the scan rate of 50 mV s−1. Thecorresponding calibration plot for pH vs. E0 (inset)

Direct electrochemistry of glucose oxidase and sensing glucose 1847

fabrication when compared to GNs/ZnO modified electrode.Graphite is cheaper and less toxic material compared to carbonnanotubes and graphene. Moreover the fabricated biosensor isdisposable one and can be prepared in less than an hour. Theseadvantages of our biosensor will gain much further attentionand can be used for real time sensing of glucose in near future.

Selectivity and practicability of the biosensor

The selectivity of the biosensor was investigated by usingcyclic voltammetry. The response of the biosensor to 2 mMglucose was checked in the presence of potential interferingbiologically active species such as dopamine (DA), ascorbic

acid (AA) and uric acid (UA). The biosensor response to2 mM glucose in the presence of 3 mM DA and AA and4 mMUA at the GNs/ZnO composite modified electrode wasanalyzed. Table S1 is evident that the potentially interferingelectro active species show less effect (<5 %) on the determi-nation of glucose. These results indicate that the fabricatedbiosensor has good selectivity even in the presence of excessconcentrations of interfering species.

To demonstrate the practical use of the fabricated biosen-sor, the glucose detection in human serum and urine sampleswere performed. The experimental conditions are same asmentioned above in the glucose electrocatalysis section.Human serum and urine sample were collected from healthymen and used without any further pretreatment except dilution

Fig. 6 a The cyclic voltammetricresponses at GOx immobilizedGNs/ZnO modified electrode inthe presence of differentconcentration of glucose inoxygen saturated pH 7 solution.bLinear dependence of [glucose]vs. Ipc

Table 1 Comparison of the analytical performance of the GOx immobilized GNs/ZnO composite modified electrode with other GOx modifiedelectrodes reported previously

Modified electrode Ks (s−1) Linear range (mM) Sensitivity (μA mM−1 cm−2) LOD (μM) Stability (%) Reference

CPE/Au/GOx 5.3 up to 0.28 8.4 10 96 (10 days) [3]

Graphene/GOx/Chi 2.83 up to 12 37.93 20 NA [24]

GCNT/GOx/GAD 1.08 up to 20.09 2.47 NA 96.3 (12 days) [29]

CNT/Au/PDDA/GOx 1.01 up to 5.2 0.25 NA 97 (6 days) [30]

ERGO-MWCNTs/GOx 3.02 up to 6.5 7.95 4.7 90 (30 days) [31]

RGO/Chi/GOx NA up to 2 NA 40 80 (30 days) [35]

MWCNT/ZnO/GOx 1.96 up to 1.29 NA 2.22 85.1 (27 days) [36]

RGO/ZnO/GOx 7.55 up to 6.6 13.7 200 80 (10 days) [37]

RGO/ZnO/GOx NA up to 27.2 18.97 20 NA [38]

Nf/Graphene/GOD 1.96 up to 14 21.9 40 NA [39]

MWCNT/ZnO/GOx 1.66 up to 27.2 4.18 20 91.3 (15 days) [40]

GNs/ZnO/GOx 3.75 up to 4.5 30.07 70 89.7 (47 days) Present study

LOD limit of detection, CPE carbon paste electrode, Au gold nanoparticles, GOx glucose oxidase, Chi chitosen, NA not available, GCNT gelatin-multiwall carbon nanotube, GAD glutaraldehyde, CNT carbon nanotube, PDDA poly (diallydimethylammonium chloride), ERGO electrochemicallyreduced graphene oxide, MWCNTs multiwalled carbon nanotubes, RGO reduced graphene oxide, ZnO zinc oxide, Nf nafion

1848 C. Karuppiah et al.

step. The glucose level in a serum sample was pre-determinedas 4.62 mM by Tecan Sunrise plate reader (http://www.tecan.com/platform/apps/product/index.asp?MenuID=1260&ID=682&Menu=1&Item=21.2.10.4). The glucose concentrationis calculated as 4.5 mM by using fabricated biosensor with aRSD of 1.9 % for two replicate measurements, indicating thegood practicality of the biosensor. To evaluate the practicalityof the biosensor in Human urine samples, the standardaddition method was used for the determination of glucose.The recovery results of glucose in urine samples aresummarized in Table S2. The good recovery results ofglucose in urine samples (∼98.3 %) authenticate the goodpracticality of the biosensor.

The storage stability of the GOx immobilized GNs/ZnOmodified electrode was examined periodically by CV. Only10.3 % of the response current is lost after 47 days indicatingthe high enzymatic activity in the biosensor. The stability ofthe GOx at GNs/ZnO composite before and after overnightdrying at room temperature was also checked by using CV.Only slight loss (0.8 %) of the redox peak current and nonotable shift in the peak potential of GOx (FAD/FADH2) areobserved, suggesting that GOx is highly stable at the GNs/ZnO composite modified electrode. The relative standarddeviation (RSD) for 7 successive determinations is 4.4 % for2 mM glucose determination. These results further indicatethat the GOx immobilized GNs/ZnO composite modifiedelectrode has good repeatability and reproducibility alongwith excellent stability.

Conclusions

In summary, the direct electrochemistry of GOx has beensuccessfully investigated at electrochemically fabricatedgraphite nanosheets (GNs) and ZnO nanoparticles (ZnO)composite modified screen printed carbon electrode (SPCE)for the first time. Surface morphological studies indicate thatGNs have more edge plane defects than pristine graphite. Thepeak current intensities of the GOx redox couple at GN/ZnOcomposite is about 2.5 and 5 folds higher than GNs/SPCE andactivated SPCE/ZnO modified electrodes. The fast and en-hanced electron transfer of GOx is attributed to the highconductivity and large edge plane defects of GNs and goodelectron communication of ZnO. The fabricated biosensorshows a lower detection limit of 70 μM with the high sensi-tivity of 30.07 μAmM−1 cm−2 for glucose determination. Thefabricated GNs/ZnO composite can bring a novel platform inprospective for the immobilization of other similar redoxactive proteins like horseradish peroxidase, cytochrome cand hemoglobin.

Acknowledgments This project was supported by the National ScienceCouncil and the Ministry of Education of Taiwan (Republic of China).

Authors express their sincere thanks to Mr. Srikanth Cheemalapati forproviding the Human blood serum samples.

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